22.5-22.6
In Pyrosequencing, how are the nucleotides flowing across the plate in sequencing?
1 at a time, when the light flashes means that the nucleotide has been incorporated
What is a metagenome?
A collection of genes from an environmental sample
Next-generation sequencing technologies
Ability to process thousands or even millions of sequence reads in parallel
PacBio SMRT: DNA Prep Enzymes Used Detection
DNA Prep - DNA fragments and DNA polymerase are trapped within tiny holes on a thin metal film Enzymes - DNA Polymerase Detection - Growth of individual DNA molecules is monitored by fluorescence imaging
Illumina: DNA Prep Enzymes Used Detection
DNA Prep - DNA fragments are bound to a flow cell surface Enzymes used - DNA Polymerase Detection - Four different fluorescently labeled nucleotides are detected
Pyrosequencing: DNA Preparation Enzymes Used Detection
DNA Prep - DNA fragments bound to small beads, which are dropped into tiny wells in a picotiter plate Enzymes used - DNA polymerase, ATP sufturylase, luciferase, apyrase Detection - Pyrophosphate release activates luciferase, which breaks down luciferin and gives off light
What was the first living organism to have its entire genome sequenced
Haemophilus influenza
Shotgun Sequencing
Method where small DNA fragments are randomly generated from larger pieces and sequenced
What is the equation for probability in shotgun sequencing?
P = e^3m P = Probability that a base will be left unsequenced e = natural log m = number of bases sequenced / total genome size
What did the scientists do the the H. influenzae fragments after breaking them into fragments?
Randomly cloned into vectors and grown in E. coli and sequenced
High-throughput sequencing
Rapidly sequencing large amounts of DNA
genome sequencing projects
Research aimed at determining the DNA sequence of the entire genome of a species
What is the most efficient and inexpensive way to sequence genomes?
Shotgun sequencing
Parallel sequencing
Simultaneously perform many sequencing runs in multiple gel-filled capillary tubes
How was the DNA from H. influenzae broken into fragments?
Sound waves
In pyrosequencing, what does the amplitude of the light indicate?
The stronger the light, the more nucleotides were added sequentially. (2x bright means 2 of the same nucleotide added AA,GG,TT,CC)
What is metagenomics?
The study of a complex mixture of genetic material from an environmental sample
What happened to the fragments after being grown in E. coli
Underwent dideoxy sequencing
During pyrosequencing, the release of pyrophosphate after addition of a nucleotide is monitored through a series of chemical reactions. In the first reaction, pyrophosphate and adenosine 5' phosphosulfate are the substrates for ATP synthesis by the enzyme ATP sulfurylase. The ATP produced in this reaction is combined with luciferin by the enzyme ________ to produce __________.
luciferase, light
In pyrosequencing, the incorporation of a nucleotide into a growing DNA strand is monitored by measuring the release of ______.
pyrophosphate (PPi)
